Unraveling the Origin of Multi-Exponential Fluorescence Intensity Decay of Tryptophan in Proteins

Abstract

The multi-exponential nature of tryptophan fluorescence intensity decay among proteins has puzzled biophysicists over all these years. Its origin has been previously attributed to, emission from multiple electronic absorption bands, namely La and Lb states; presence of a mixture of Trp rotamers that possess unique fluorescence lifetimes or multiple conformational states of the protein. However, none of the above explanations have been proved conclusive. Here, we report that the multi-exponential decay originates due to additional luminescence contribution from protein charge transfer states. We have previously shown (Chem. Sci., 2017, 8, 5416) the existence of weak but significant electronic absorption between 250—800 nm among proteins rich in charged residues resulting in Protein Charge Transfer Spectra (ProCharTS). Separated charges in the protein excited state created by photoinduced electron transfer emit weak luminescence on charge recombination. We observed that fluorescence intensity decay of N-Acetyl-L-Tryptophanamide (NATA) on excitation at 295 nm showed gradual deviation from mono-exponential decay kinetics with increasing presence of different charged amino acids (D, E, K, H and R). Notably contribution from a ∼2 ns component showed a significant increase in presence of charged amino acids at expense of ∼3 ns component arising from indole. The presence of charged amino acids also quenched its fluorescence lifetime (from ∼3 to ∼ 2.3 ns). Further, influence of ProCharTS was not restricted to intrinsic fluorophore like Trp. A charge rich protein like human serum albumin on conjugation with Dansyl (2-dimethyl amino naphthalene 6-sulphonyl chloride) probe displays a low mean fluorescence lifetime of ∼6.5 ns, while the same probe on conjugation to relatively less charge rich protein like hen lysozyme displays a lifetime of ∼14 ns. Thus, ProCharTS emission has a significant role in origin of multi‒exponential decay kinetics of Trp in proteins.